越来越多的证据表明，父辈在环境暴露、营养状况等改变下，某些获得性性状可通过精子遗传给下一代。然而，目前对该现象背后的具体表观遗传信息载体尚缺乏清晰的认识。申请人实验室近期在哺乳动物成熟精子和血清中发现了一类进化上保守、来源于tRNA5’端序列、且高度富集的新型小RNA：tsRNAs；并进一步在高脂饮食诱导的父代肥胖小鼠模型中，发现tsRNAs及其携带的RNA修饰m5C可为一种表观遗传信息的载体，将高脂诱导的父代代谢紊乱表型传递给子代 (Science, 2016)。然而，精子tsRNAs在调节早期胚胎发育中的靶基因是什么？精子tsRNAs上的修饰m5C受何种酶调控并参与跨代信息的传递？本课题将针对上述两个问题，通过RNA测序，受精卵RNA注射，RNA修饰谱差异筛选、以及RNA修饰酶DNMT2敲除小鼠等多种方法和模型，力争对精子tsRNAs调控获得性性状的跨代遗传的分子机理提供更深入的认识。

Sperm RNA mediated transmission of phenotypes has drawn increasing attention as an important type of non-genetic (epigenetic) inheritance. However, the exact carrier of such RNA-mediated information remains elusive and remains the open question in the field. By analyzing small RNA deep-sequencing data, we recently discovered that the mammalian mature sperm contain a unique subset of highly enriched small RNA families derived from 5’ halves of mature tRNAs, mostly being 29-34nt in length, which are named tsRNAs (tRNA-derived small RNAs). In a paternal mouse model given a high-fat diet (HFD), we showed that sperm tsRNAs exhibited changes in expression profiles and RNA modifications. Injection of sperm tsRNA fractions from HFD males into normal zygotes generated metabolic disorders in the F1 offspring, demonstrating that sperm tsRNAs represent a paternal epigenetic factor that may mediate intergenerational inheritance of diet-induced metabolic disorders. These discoveries raise the open question that how sperm tsRNAs, along with their RNA modifications, mediate the embryonic developmental programming that affect the offspring phenotype, thus are the emphases of this present project. Here, we propose to (1) test sperm tsRNAs’ target by injecting sperm tsRNAs (from HFD and control male) into zygote, followed by comparative single-cell embryo RNA-seq of 2- to 4-cell embryos and bioinformatics analysis. (2) Because significant alteration of m5C (5-methylcytidine) occurs in sperm tsRNAs from HFD males, we will further test whether the m5C cytosine RNA methyltransferase, DNMT2, is essential for sperm tsRNAs’ ability to transmit acquired metabolic disorder to offspring. Utilizing the HFD model in Dnmt2-/- and Dnmt2+/+ male mice, we will inject sperm tsRNAs into normal zygotes and examine the metabolic phenotype of F1 offspring. The overall aim of present project is to determine the mechanisms by which sperm tsRNAs establish transcriptional changes in early embryos that lead to offspring phenotype and to identify the RNA modification (and the enzyme) essential for transmitting the paternally acquired phenotype.